2.4 Determination of microorganisms
The 0.4 g sediment samples were
taken at the final sampling (2022.8.4) and stored at -80 °C for DNA
extraction. Sediment DNA was extracted using a Fast DNA Spin Kit for
Soil (Qbiogene Inc., Carlsbad, CA, USA) according to the manufacturer’s
instruction. The extracted DNA was used as template for PCR
amplification. The 20 μL PCR
reaction system contained 4 μL of 5×FastPfu Buffer, 2 μL of dNTPs (2.5
mmol L-1), 0.4 μL of FastPfu Polymerase, 0.8
μL of each primer (5 μmol
L-1), 0.2 μL of BSA, 1 μL of template DNA, and 10.8 μL
of ddH2O. In order to investigate the dynamic variations
of the microbial community structure, DNA samples were PCR amplified
with the primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R
(5′-GACTACHVGGGTATCTAATCC-3′) (Herlemann et al. 2011) and pyrosequenced
using a HiSeq sequencer of Illumina
by the standard PCR methods and protocols. PCR was performed as follows:
initial denaturation at 95 °C for 3 min, followed by 30 cycles of
denaturation at 95 °C for 30 s, annealing at 53 °C for 30 s, and
extension at 72 °C for 45 s. The amplified products were detected by 2
% agar gel electrophoresis. The purified amplicons were mixed in
equimolar amounts and sequenced on an Illumina Hiseq2500 PE250 platform.